Supplementary Table 1 Sequences of the primer used for q-PCR analysis

Slides:

Advertisements

Similar presentations

FIGURE S1 S1.c Scr. iPARG 30 nM H2O2 PAR iPARP-1 60 nM DAPI S1.a S1.b

Advertisements

Comprehensive Analysis of the Chemical Composition and In Vitro Cytotoxic Mechanisms of Pallines Spinosa Flower and Leaf Essential.

Aldehyde Dehydrogenase 1A1 Possesses Stem-Like Properties and Predicts Lung Cancer Patient Outcome Xiao Li, MD, Liyan Wan, MD, Jian Geng, MD, Chin-Lee.

Article title: PI3K independent activation of mTORC1 as a target in lapatinib-resistant ERBB2+ breast cancer cells Journal: Breast Cancer Research and.

STAT3 mediates oncogenic addiction to TEL-AML1 in t(12;21) acute lymphoblastic leukemia by Maurizio Mangolini, Jasper de Boer, Vanessa Walf-Vorderwülbecke,

Volume 145, Issue 2, Pages (August 2013)

Tumor-Derived Jagged1 Promotes Osteolytic Bone Metastasis of Breast Cancer by Engaging Notch Signaling in Bone Cells Nilay Sethi, Xudong Dai, Christopher.

Antiangiogenic antithrombin down-regulates the expression of the proangiogenic heparan sulfate proteoglycan, perlecan, in endothelial cells by Weiqing.

Crucial Roles of MZF1 and Sp1 in the Transcriptional Regulation of the Peptidylarginine Deiminase Type I Gene (PADI1) in Human Keratinocytes Sijun Dong,

Volume 19, Issue 7, Pages (July 2017)

DNMT3B Overexpression by Deregulation of FOXO3a-Mediated Transcription Repression and MDM2 Overexpression in Lung Cancer Yi-Chieh Yang, MS, Yen-An Tang,

Sp1 Suppresses miR-3178 to Promote the Metastasis Invasion Cascade via Upregulation of TRIOBP Hui Wang, Kai Li, Yu Mei, Xuemei Huang, Zhenglin Li, Qingzhu.

Volume 17, Issue 2, Pages (October 2016)

The C/EBPδ tumor suppressor is silenced by hypermethylation in acute myeloid leukemia by Shuchi Agrawal, Wolf-Karsten Hofmann, Nicola Tidow, Mathias Ehrich,

Volume 55, Issue 1, Pages (July 2014)

by Christopher J. Ott, Nadja Kopp, Liat Bird, Ronald M

A B Untreated MM1 15 mM GRN163L 15mM

Volume 28, Issue 5, Pages (November 2015)

Cell Physiol Biochem 2016;39: DOI: /

Volume 118, Issue 4, Pages (April 2000)

Inhibition of glycogen synthase kinase-3 activity leads to epigenetic silencing of nuclear factor κB target genes and induction of apoptosis in chronic.

MicroRNA-381 Represses ID1 and is Deregulated in Lung Adenocarcinoma

NF1 silencing confers resistance of PC9 lung adenocarcinoma cells to erlotinib (erl). NF1 silencing confers resistance of PC9 lung adenocarcinoma cells.

PPARγ signaling correlates with LATS2 in human and mouse tumors and promotes cell death in a LATS2-dependent manner. PPARγ signaling correlates with LATS2.

Volume 16, Issue 9, Pages (August 2016)

Aldehyde Dehydrogenase 1A1 Possesses Stem-Like Properties and Predicts Lung Cancer Patient Outcome Xiao Li, MD, Liyan Wan, MD, Jian Geng, MD, Chin-Lee.

Molecular Therapy - Nucleic Acids

Volume 30, Issue 1, Pages (April 2008)

Volume 2, Issue 5, Pages (November 2002)

Yuen Yee Cheng, PhD, Michaela B

Volume 20, Issue 2, Pages (February 2018)

SUPPLEMENTAL TABLE 1. Cell cycle profiles of HeLa cells treated

Volume 5, Issue 6, Pages (December 2013)

Volume 138, Issue 5, Pages e2 (May 2010)

Activation of the Innate Signaling Molecule MAVS by Bunyavirus Infection Upregulates the Adaptor Protein SARM1, Leading to Neuronal Death Piyali Mukherjee,

Uc.454 Inhibited Growth by Targeting Heat Shock Protein Family A Member 12B in Non- Small-Cell Lung Cancer Jun Zhou, Chenghai Wang, Weijuan Gong, Yandan.

Volume 145, Issue 2, Pages (August 2013)

Molecular Therapy - Nucleic Acids

Volume 11, Issue 2, Pages (February 2007)

CHK1 downregulation upon ERG overexpression.

Volume 13, Issue 10, Pages (December 2015)

Volume 25, Issue 3, Pages (March 2017)

Brian Poligone, Elaine S. Gilmore, Carolina V

LATS2 promotes death of lumB cells.

Regulation of α-Synuclein Expression by Poly (ADP Ribose) Polymerase-1 (PARP-1) Binding to the NACP-Rep1 Polymorphic Site Upstream of the SNCA Gene Ornit.

Andrew J Henderson, Ruth I Connor, Kathryn L Calame Immunity

PARP1 Represses PAP and Inhibits Polyadenylation during Heat Shock

Minchul Kim, Taekhoon Kim, Randy L. Johnson, Dae-Sik Lim Cell Reports

Jungmook Lyu, Vicky Yamamoto, Wange Lu Developmental Cell

Supplementary Figure 1. Structure of amlexanox

Volume 4, Issue 3, Pages (March 2015)

Nucleocapsid Phosphorylation and RNA Helicase DDX1 Recruitment Enables Coronavirus Transition from Discontinuous to Continuous Transcription Chia-Hsin.

Volume 20, Issue 4, Pages (October 2011)

Volume 13, Issue 4, Pages (April 2013)

N H N H Figure S1 parp-1 +/+ parp-1 -/- HIF-2 DAPI

Volume 10, Issue 3, Pages (September 2006)

Figure 1. RSPO3 expression is upregulated in bladder cancer

Volume 127, Issue 4, Pages (October 2004)

Shrimp miR-34 from Shrimp Stress Response to Virus Infection Suppresses Tumorigenesis of Breast Cancer Yalei Cui, Xiaoyuan Yang, Xiaobo Zhang Molecular.

MicroRNA-381 Represses ID1 and is Deregulated in Lung Adenocarcinoma

Volume 26, Issue 3, Pages (May 2007)

Long Noncoding RNA BC as a Novel Therapeutic Target for Colorectal Cancer that Suppresses Metastasis by Upregulating TIMP3 Jiaxin Lin, Xin Tan,

MELK Promotes Melanoma Growth by Stimulating the NF-κB Pathway

Volume 18, Issue 3, Pages (March 2010)

Volume 49, Issue 5, Pages (March 2013)

Volume 5, Issue 6, Pages (December 2013)

Shipra Das, Olga Anczuków, Martin Akerman, Adrian R. Krainer

Molecular Therapy - Nucleic Acids

Volume 55, Issue 1, Pages (July 2014)

Fig. 2 hTERT induces expression of heat shock protein genes through HSF1 and interacts with Hsp70-1. hTERT induces expression of heat shock protein genes.

Presentation transcript:

Supplementary Table 1 Sequences of the primer used for q-PCR analysis hGAPDH forward, 5’-AGCAATGCCTCCTGCACCACCAAC-3’ hGAPDH reverse, 5’-CCGGAGGGGCCATCCACAGTCT-3’ hPARP1 forward, 5’-TCTACCCCCTGGAGATTGACT-3’ hPARP1 reverse, 5’-GGTCGATCTCATACTCCACCAT-3’ hPARP3 forward, 5’-AACTGGGTAATCGGAAGCTG-3' hPARP3 reverse, 5'-ACCAGAATGTGGCATGATGC-3' hPARG forward, 5'-AGCAAGAGACGCTGACATTG-3' hPARG reverse, 5'-TTCCAGTGCGACTCTCTCCT-3'

A Low risk High risk 2log PARP1 p 1.0e-06 n=318 n=83 6.0 8.5 8.0 7.5 7.0 6.5 MNSC B C 1.0 0.8 0.6 0.4 0.2 0.0 48 96 144 192 Overall survival Follow up in months High PARP1 (n=101) LowPARP1 (n=300) p 9.4e-07 MNSC 1.0 0.8 0.6 0.4 0.2 0.0 48 96 144 192 Follow up in months Event-free survival High PARP1 (n=101) LowPARP1 (n=300) MNSC p 7.8e-06 D E 1.0 0.8 0.6 0.4 0.2 0.0 Overall survival 48 96 144 192 Follow up in months p 1.7e-03 High PARP1 (n=82) LowPARP1 (n=240) Low Risk 1.0 0.8 0.6 0.4 0.2 0.0 Event-free survival 48 96 144 192 Follow up in months p 1.1e-03 High PARP1 (n=82) LowPARP1 (n=240) Low Risk F G 1.0 0.8 0.6 0.4 0.2 0.0 Overall survival 48 96 144 192 Follow up in months p 7.0e-04 High PARP1 (n=20) LowPARP1 (n=63) MNSC/High Risk 1.0 0.8 0.6 0.4 0.2 0.0 48 96 144 192 Follow up in months p 1.6e-03 High PARP1 (n=20) LowPARP1 (n=63) MNSC/High Risk Event-free survival Figure S1 PARP1 expression predicts poor outcome in neuroblastoma patients. A, Box plot of PARP1 expression relative to risk. B, C, D, E, F, G, Kaplan-Meier curves reporting patients overall and event-free survival probability with respect to PARP1 expression in the MNSC (B, C), low risk (D, E) and MNSC/high risk (F, G) patients subgroups. P value as indicated.

A B 2log PARP2 3.5 6.0 5.5 5.0 4.5 4.0 Stage 1 Stage 2 Stage 3 Stage 4 Stage 4S n=53 n=183 n=121 n=78 n=63 MNSC MNA p 1.5e-04 n=401 n=92 2log PARP2 3.5 6.0 5.5 5.0 4.5 4.0 C D Low Risk High Risk p 5.1e-12 n=322 n=176 2log PARP2 3.5 6.0 5.5 5.0 4.5 4.0 Low Risk High Risk p 3.3e-08 n=318 n=83 2log PARP2 3.5 6.0 5.5 5.0 4.5 4.0 MNSC 6.5 E F 1.0 0.8 0.6 0.4 0.2 0.0 Overall survival High PARP2 (n=126) LowPARP2 (n=372) 48 96 144 192 Follow up in months p 1.0e-03 1.0 0.8 0.6 0.4 0.2 0.0 Event-free survival High PARP2 (n=126) LowPARP2 (n=372) 48 96 144 192 Follow up in months p 5.9e-03 Figure S2 PARP2 expression, like PARP1, predicts poor prognosis and is associated with stage, MYCN status and risk in NB patients. A, B, C, D, Box plot of PARP2 expression relative to tumor stage (A), MYCN status (B), and risk classification in the total set (C) or in the MNSC subgroup (D). P values: 5.2e-03 (stage 2 versus stage 4) and 0.02 (stage 4 versus stage 4S). E, F, Overall (E) and event-free (F) survival relative to PARP2 expression in NB patients. P value as indicated.

mRNA expression (fold change) B C MNSC MNA Relative PARP2 mRNA expression 0.00 0.02 0.04 0.06 0.08 0.0 0.1 0.2 mRNA expression Relative b-Actin PARP1 MYCN SK-N-SH SK-N-MYC * Genes MNSC MNA p value PARP1 7,38 4.90E-13 PARP2 4,85 1.50E-04 PARP3 0.06 PARP4 0.15 PARP5a 6,95 2.50E-03 PARP5b 6,14 6.00E-09 PARP6 7,09 4.90E-05 PARP7 3 6.50E-03 PARP8 4,75 8.30E-12 PARP9 3,11 1.10E-03 PARP10 4,41 3.10E-11 PARP11 0.27 PARP12 4,08 1.80E-15 PARP13 1.30E-08 PARP14 2.10E-10 PARP15 9.10E-09 PARP16 4,14 7.60E-48 PARG 4,53 2.80E-08 ARH3 4,43 2.50E-47 TARG1 6,91 5.20E-24 D * *** PARG PARP3 mRNA expression (fold change) 2.5 2.0 1.0 0.5 1.5 MYCN+ MYCN- Color key: Low High expression Figure S3 PARP isoforms expression in neuroblastoma. A, Heat map diagram of PARP isoforms and PAR degrading enzymes (PARG, TARG1 and ARH3) expression in primary neuroblastoma samples (GSE62564 R2-dataset). Samples are clustered by MYCN status (MNSC, MYCN single copy and MNA, MYCN amplified). Expression levels are indicated by color intensity as shown in the color key legend. P values are reported. B, Real Time q-PCR of PARP2 expression in the MNSC and MNA cell lines reported in Fig. 1. C, Real Time q-PCR and WB analysis of PARP1 expression in the stably transfected SK-N-MYC compared with the control. For qPCRs, the levels of PARP1 and PARP2 mRNA were normalized on GAPDH expression. Data are reported as averages (+/- SD) of two independent experiments (*P<0.05). D, Real time q-PCR of PARG and PARP3 expression in the MYCN+/- SHEP Tet21/N system confirms R2-based expectations. The levels of mRNA were normalized on GAPDH expression. Data are reported as averages (+/- SD) of three (for PARG) and two (for PARP3) independent experiments (* P<0.05; ***P<0.005).

A B PAR Hsp70 Ola + - SK-N-SH Kelly MYCN- MYCN+ 1.0 1.2 0.6 0.8 0.4 0.2 Colony number (%) Kelly SK-N-SH 40 20 80 60 100 120 CTR 5 mM 10 mM ** C D Colony number (%) CTR 5 mM 10 mM MYCN- MYCN+ ** Time (hours) Cell number (*10^4) MYCN- CTR MYCN+ CTR MYCN- Ola MYCN+ Ola 48 72 96 Figure S4 MNA and MYCN-OE cells are more sensitive to PARP inhibitors. A, WB analysis of PARylation in olaparib treated MNSC, MNA and MYCN-overexpressing cells. Blots were probed with anti-PAR antibody and Hsp70 was used as loading control. B, C Colony formation assay performed on SK-N-SH, Kelly (B) and MYCN+/- (C) neuroblastoma cells after exposure to olaparib or DMSO (CTR) for 24 hours. Data are reported as averages (+/- SD; **P<0.01). D, Viable cell counts performed on MYCN+/- cells treated with or without olaparib for the indicated times. Data are reported as averages (+/- SD) of three independent experiments.

A B C D E 7-AAD Figure S5 Olaparib effects on neuroblastoma cells. Kelly CTR SK-N-SH CTR Kelly Ola SK-N-SH Ola 7-AAD EdU # cells C D Ola - + SK-N-SH Kelly Tail moment (fold ind.) 24 h 15 h *** + Kelly Ola p53S15P p53 Chk1S345P Chk1 Cdc25a b-actin - SK-N-SH 3h 6h 9h 12h 24h E EdU + cells (%) 9 12 24 MYCN+/CTR MYCN-/CTR MYCN+/Ola MYCN-/Ola Figure S5 Olaparib effects on neuroblastoma cells. A, Flow cytometry analysis of DNA synthesis (EdU) and DNA content (7-AAD) in indicated cells treated with or without olaparib for 48 hours. Cell cycle profiles are reported in lower panels. The arrowhead indicates the EdU-negative nuclei featuring <4N DNA content. B, Representative images of nuclear morphology in Hoechst 33258 stained MYCN+/- cells treated with or without olaparib for 48 hours. (Scale bar: 10μm). C, Neutral comet assay performed in the indicated cells treated with or without olaparib for 15 and 24 hours. Data are reported as averages (+/- SE) of three independent experiments (*** P<0.001). D, WB analysis of whole cell lysates prepared from the indicated cells. Blots were probed with the indicated antibodies. E, Percentage of EdU positive cells in MYCN+/- treated with or without olaparib at indicated times.

A B C 1 10 Ola [mM] Cell cycle distribution (%) Ola [mM] 0.01 0.1 1 10 PAR b-actin CHKSer345P CHK1 Ola [mM] 1 10 b-actin D 0.01 0.1 1 10 0.5 2 PAR b-actin Vel [mM] Tal [mM] Figure S6 PARP enzymatic inhibition is not sufficient to induce cell cycle dysregulation. A, WB analysis of PARylation in MYCN-overexpressing cells after 9 hours of olaparib treatment at the indicated concentrations. Blots were probed with anti-PAR antibody and b-actin was used as loading control. MYCN-overexpressing cells treated for 9 hours with or without olaparib at the indicated concentrations were analyzed by flow cytometry for DNA content (B) and by WB for CHK1 phosphorylation (C). D, WB analysis of PARylation in MYCN-overexpressing cells after 48 hours of veliparib (Vel) and talazoparib (Tal) treatment at indicated concentrations. Blots were probed with anti-PAR antibody and anti b-actin as loading control.